Abstract:
“DNA barcoding” is a powerful tool for species identification and used in studying
evolutionary relationships among species. The objective of this study was to characterize
Cytochrome c Oxidase subunitI (COI) and Internal Transcribed Spacer 2 (ITS2) sequences
of morphologically identified Anopheles species collected from two different areas in Sri
Lanka and to distinguish sibling species using DNA barcodes.
Anopheles peditaneatus, An. nigerrimus, An. jamseii, An. tessellatus, An. annularis, An.
pallidus, An. barbirostris, An. vagus, An. subpictus, An. culicifacies and An. varuna were
collected from Jaffna in the Northern (N) Province and Wariyapola in the North Western
(NW) Province. COI sequences (52) of 3,3,7,1,2,6,1,6,8,8, and 6 and, ITS2 sequences (21)
4,3,6,0, 1,5,0,2,0,0,0 were respectively generated from the PCR positive samples of DNA
extracted from the above species (10 individuals from each species).The COI sequences
showed high genetic variation compared to that of ITS2. Two of the morphologically
identified An. peditaneatus specimens had sequences similar to that of An. nigerrimus
which show high resemblance in morphological features to An. peditaneatus. Based on
COI sequences, An. subpictus samples from N Province were identified as An. subpictus
B and samples of An. subpictus from NW Province had sequences similar to sibling species
A. Analysis of COI and ITS2 sequences confirmed the presence of An. annularis sibling
species A in the N Province. An. barbirostris sequence obtained from the N Province was
different from the previously described sequences of An. barbirostris complex suggesting
the existence of a different molecular type. Sequences of morphologically identified other
anopheline species confirmed their identity with GenBank entries from South and
Southeast Asian countries. However cytologically identified sibling species B and E of An.
culicifacies could not be separated using COI and ITS2 markers. The present study
indicates that DNA barcoding is useful in identifying sibling species and proves that Sri
Lankan Anopheles species have a close evolutionary relationship with the South and
Southeast Asian counterparts.
